1. The Field of the Invention
The present invention is directed to the field of thermocyclers used in the practice of the polymerase chain reaction (PCR).
2. The Relevant Technology
An important tool in the field of molecular biology is the process known as the “polymerase chain reaction” (PCR). PCR generates large quantities of genetic material from small samples of the genetic material.
The PCR process is performed in a small reaction vial containing components for DNA duplication: the DNA to be duplicated, and PCR reaction agents that include the four nucleotides which are assembled to form DNA, two different types of synthetic DNA called “primers” (one for each of the complementary strands of DNA), and an enzyme called DNA polymerase, typically entrained in a carrier fluid.
DNA is double stranded. The PCR process begins by separating the two strands of DNA into individual complementary strands, a step which is referred to as “denaturation.” This is typically accomplished by heating the PCR reaction mixture to a temperature of about 94 to about 96 degrees centigrade for a period of time between a few seconds to over a minute in duration.
Once the DNA is separated into single strands, the mixture is cooled to about 45 to about 60 degrees centigrade (typically chosen to be about 5 degrees below the temperature at which the primer will melt) in order to allow a primer to bind to each of the corresponding single strands of DNA in the mixture. This step is typically called “annealing.” The annealing step typically takes anywhere from a few seconds up to a few minutes.
Next, the reaction vessel is heated to about 72 to 73 degrees centigrade, a temperature at which DNA polymerase in the reaction mixture acts to build a second strand of DNA onto the single strand by adding nucleic acids onto the primer so as to form a double stranded DNA that is identical to that of the original strand of DNA. This step is generally called “extension.” The extension step generally takes from a few seconds to a couple minutes to complete.
This series of three steps, also sometimes referred to as “stages”, define one “cycle.” Completion of a PCR cycle results in doubling the amount of DNA in the reaction vial. Repeating a cycle results in another doubling of the amount of DNA in the reaction vial. Typically, the process is repeated many times, e.g. 10 to 40 times, resulting in a large number of identical pieces of DNA. Performing 20 cycles results in more than a million copies of the original DNA sample. Performing 30 cycles results in more than a billion copies of the original DNA sample. A “thermocycler” is used to automate the process of moving the reaction vessel between the desired temperatures for the desired period of time.
DNA for amplification must be obtained from a biological sample, which involves disrupting biological tissues, cell walls, capsids, or the like, in order to release a particular DNA of interest. In some instances, a particular RNA is of interest, although the RNA must be converted to DNA in order to use PCR for amplification.
A challenge in managing PCR reactions occurs when the temperature range at which denaturation takes places is near the boiling temperature of the carrier fluid. For example, at higher altitudes such as might be found in cities like Denver, water, a typical carrier fluid, boils at 97 degrees centigrade. Heating a sample from about 94 to about 96 degrees centigrade may cause bubbles of water vapor and/or dissolved gases to form. These bubbles pose a challenge during PCR, particularly those systems that rely upon optical detection systems. That is, optical detection systems project an optical signal through the reaction vessel and the sample. Bubbles, however, can at least partially occlude and/or distort and/or refract the optical signal and, in so doing, degrade the signal quality and/or introduce noise received at the optical detector. Prior art solutions attempted to resolve this problem by using very precise and, consequently, very expensive thermal controls to try and prevent boiling from occurring, often with limited success.
Bubbles may also be present before the thermocycling process, such as those that develop in what can be turbulent flow of the sample as it flows into the reaction vessel or pockets of gas that become trapped within the reaction vessel when the sample enters the reaction vessel. Such bubbles pose the same problems as those generated during thermocycling.
In addition, the presence and development of bubbles within the fluid alters the volume of the fluid. This occurs as the volume of the bubbles change much more significantly than the volume of the fluid and sample change during thermocycling. The change in the volume of the bubbles cause the fluid and entrained DNA sample to flow in and out of the reaction vessel. Such flows of the fluid in and out of the reaction vessel may alter and/or dilute the concentration of the DNA sample and/or the PCR reaction agents within the reaction vessel.
Thus, there exists a need for a PCR process and system that improves signal quality. In addition, there exists a need for a PCR system that reduces the presence of bubbles within the sample.